中国医科大学学报2018，Vol.47Issue(2)：102-106,5.DOI:10.12007/j.issn.02584646.2018.02.002
细菌人工染色体技术在人巨细胞病毒基因组小片段序列置换突变中的应用
Application of Bacterial Artificial Chromosome in Small Fragment Mutation of the Human Cytomegalovirus Genome
摘要
Abstract
Objective To evaluate the applicability of Han strain bacterial artificial chromosome (Han-BAC) in small fragment mutation of the human cytomegalovirus (HCMV) genome. Methods A 31-bp long fragment of LUNA between UL80 and UL82 in the HCMV was chosen as the mutation target. Kanamycin resistance gene sequence flanking the homologous arms of the target neighbor sequence was used to replace the target sequence. Electronic transformation was used to rescue the mutated virus. Reverse transcription PCR and cDNA clone sequencing were used to identify the mRNA expression and the 3' terminal structures of LUNA,UL80,and UL82 transcripts. Results The 31 bp fragment was replaced precisely by the kanamycin resistance gene sequence. The efficiency of mutation was more than 3％. LUNA-mutated virus was rescued successfully. Transcriptions and 3' terminal structures of the UL80 and UL82 transcripts of the mutant virus were the same as those of its original virus. Conclusion The sequence at the transcription start site of LUNA was replaced successfully. The HCMV Han-BAC supports fragment mutation as small as 31 bp with a relatively high efficiency.关键词
人巨细胞病毒/细菌人工染色体/LUNA/突变Key words
cytomegalovirus/bacterial artificial chromosome/LUNA/mutation分类
医药卫生引用本文复制引用
柳中洋,卢颖,韩丽英,马艳萍,齐莹,黄郁晶,阮强..细菌人工染色体技术在人巨细胞病毒基因组小片段序列置换突变中的应用[J].中国医科大学学报,2018,47(2):102-106,5.基金项目
国家自然科学基金 (81371788) （81371788）
